Synthesis And Biological Evaluation Of Novel Stilbene Analogues And Their Application In Shrimp Preservation

Penaeus vannamei,is rich in nutrition,delicious,thus is a kind high-quality seafood.However,due to the high protein and water contents,shrimp is affected easily by bacteria,polyphenoloxidase and other factors during the storage,resulting in the blackening and spoilage,and the decline of its nutritional and economic value.At present,"Everfresh"(4-hexyl resorcinol is the active component)is the most effective shrimp preservative,but its application is still limited by its high cost.Therefore,it is significant to develop a shrimp preservative with high efficiency,safety and low cost.In this thesis,a series of novel stilbene analogues were ynthesized using kojic acid as a starting material,and their inhibitory activity and kinetics against mushroom tyrosinase were determined.The antioxidant and bacteriostatic abilities of the stilbene analogues 6c and 8h were studied,and the bacteriostatic mechanism of the compounds was further explored.Finally,the compound 8h was applied to the preservation of Penaeus vannamei,and its residue in shrimp samples was determined.The results of this thesis provide a theoretical basis for the development of new shrimp preservatives with independent intellectual property rights.The main results of the test are as follows:1.A series of novel stilbene analogues 6 and 8 were designed and synthesized starting from kojic acid.Their structures were confirmed by 1H NMR,13C NMR,ESI-MS and ESI-HRMS.2.The inhibitory activities on tyrosinase monophenolase,diphenolase and inhibitory kinetics of compounds 6 and 8 were investigated.The results showed that compound 6c and 8h possessed the strongest inhibitory effects on monophenolase of tyrosinase with the IC50 of 1.56 and 2.72μM,respectively.The inhibitory activity of 6c and 8h was 8.03 and 4.60 times that of kojic acid.The IC50 of the compound 6c and 8h on diphenolase was determined as 7.15 and 15.86 μM,respectively.The results showed that the inhibition of the compounds 6c and 8h on tyrosinase was reversible and of a competitive-uncompetitive mixed type.The inhibition constants of 6c and 8h against free enzyme(KI)were determined as 14.03 and 40.90 μM,respectively,and the data for enzyme-substrate complex(KIS)being 21.86 and 50.25 μM,respectively.The mechanism of tyrosinase inhibition was analyzed by determining the reducing and chelating ability of compounds 6c and 8h to Cu2+.3.The results of in vitro antioxidant activity test showed that the compounds 6c and 8h are able to scavenge DPPH radical,superoxide anion free radical(O2-·)and hydroxyl free radical(OH),and their antioxidant effect was stronger than that of kojic acid(P<0.05).4.The results of dominant spoilage bacteria screening test showed that Shewanella amazonensis was the dominant spoilage bacterium among the six bacterial strains isolated from Penaeus vannamei.The bacteriostatic effect and mechanism of the compounds 6c and 8h were studied by determination of minimum inhibitory concentration(MIC),minimum bactericidal concentration(MBC)and micromorphology assays of bacteria and so on.The results showed that the bacteriostatic effect of these two compounds was significantly stronger than that of kojic acid,and the bacteriostatic ability of 8h was stronger than that of 6c.In the aspect of bacteriostatic mechanism,the results showed that compounds 6c and 8h could inhibit the growth of bacteria by destroying the cell membrane structure of the bacteria,and could make the bacteria obviously distort and collapse by changing the external environment of the bacteria.They also acted as bacteriostatic agents by chelating the iron ions,which is essential for the bacterial growth.Moreover,the strongest synergistic bacteriostatic effect was observed by the use of compound 8h in combination with citric acid.5.The effect of inhibitor 8h on the freshness of Penaeus vannamei was studied by determining the changes in weight loss rate and pH value of the shrimp samples and so on.The synergistic preservation effect of 8h combined with ascorbic acid or citric acid was investigated.The results showed that the fresh-keeping effect of the 8h treatment group was significantly better than that of the control group,and was better than that of the kojic acid treatment group and the 4-HR treatment group,and the shelf life(at 4 ℃)of the shrimp was extended to 10 days.In addition,the formula 8h+CA was found to possess the best preservation effect via further inhibiting the changes in pH,TVB-N,TBA and texture of shrimp samples,thus extending the shelf life(4 ℃)of Penaeus vannamei to 12 days.6.The results of 8h residue determination showed that the residues of compound 8h in shrimp meat samples were very low.8h was mainly adhered to the surface of shrimp carapace and a few existed in shrimp meat.The residues of 8h in shrimp carapace and shrimp meat decreased with the prolongation of storage time.